Hsa-LINC02418/mmu-4930573I07Rik regulated by METTL3 dictates anti-PD-L1 immunotherapeutic efficacy via enhancement of Trim21-mediated PD-L1 ubiquitination.

BACKGROUND: Limited response to programmed death ligand-1 (PD-L1)/programmed death 1 (PD-1) immunotherapy is a major hindrance of checkpoint immunotherapy in non-small cell lung cancer (NSCLC). The abundance of PD-L1 on the tumor cell surface is crucial for the responsiveness of PD-1/PD-L1 immunotherapy. However, the negative control of PD-L1 expression and the physiological significance of the PD-L1 inhibition in NSCLC immunotherapy remain obscure. METHODS: Bioinformatics analysis was performed to profile and investigate the long non-coding RNAs that negatively correlated with PD-L1 expression and positively correlated with CD8+T cell infiltration in NSCLC. Immunofluorescence, in vitro PD-1 binding assay, T cell-induced apoptosis assays and in vivo syngeneic mouse models were used to investigate the functional roles of LINC02418 and mmu-4930573I07Rik in regulating anti-PD-L1 therapeutic efficacy in NSCLC. The molecular mechanism of LINC02418-enhanced PD-L1 downregulation was explored by immunoprecipitation, RNA immunoprecipitation (RIP), and ubiquitination assays. RIP, luciferase reporter, and messenger RNA degradation assays were used to investigate the m6A modification of LINC02418 or mmu-4930573I07Rik expression. Bioinformatics analysis and immunohistochemistry (IHC) verification were performed to determine the significance of LINC02418, PD-L1 expression and CD8+T cell infiltration. RESULTS: LINC02418 is a negative regulator of PD-L1 expression that positively correlated with CD8+T cell infiltration, predicting favorable clinical outcomes for patients with NSCLC. LINC02418 downregulates PD-L1 expression by enhancing PD-L1 ubiquitination mediated by E3 ligase Trim21. Both hsa-LINC02418 and mmu-4930573I07Rik (its homologous RNA in mice) regulate PD-L1 therapeutic efficacy in NSCLC via Trim21, inducing T cell-induced apoptosis in vitro and in vivo. Furthermore, METTL3 inhibition via N6-methyladenosine (m6A) modification mediated by YTHDF2 reader upregulates hsa-LINC02418 and mmu-4930573I07Rik. In patients with NSCLC, LINC02418 expression is inversely correlated with PD-L1 expression and positively correlated with CD8+T infiltration. CONCLUSION: LINC02418 functions as a negative regulator of PD-L1 expression in NSCLC cells by promoting the degradation of PD-L1 through the ubiquitin-proteasome pathway. The expression of LINC02418 is regulated by METTL3/YTHDF2-mediated m6A modification. This study illuminates the underlying mechanisms of PD-L1 negative regulation and presents a promising target for improving the effectiveness of anti-PD-L1 therapy in NSCLC.

SLC25A38 as a novel biomarker for metastasis and clinical outcome in uveal melanoma.

Risk of metastasis is increased by the presence of chromosome 3 monosomy in uveal melanoma (UM). This study aimed to identify more accurate biomarker for risk of metastasis in UM. A total of 80 patients with UM from TCGA were assigned to two groups based on the metastatic status, and bioinformatic analyses were performed to search for critical genes for risk of metastasis. SLC25A38, located on chromosome 3, was the dominant downregulated gene in metastatic UM patients. Low expression of SLC25A38 was an independent predictive and prognostic factor in UM. The predictive potential of SLC25A38 expression was superior to that of pervious reported biomarkers in both TCGA cohort and GSE22138 cohort. Subsequently, its role in promoting metastasis was explored in vitro and in vivo. Knock-out of SLC25A38 could enhance the migration ability of UM cells, and promote distant metastasis in mice models. Through the inhibition of CBP/HIF-mediated pathway followed by the suppression of pro-angiogenic factors, SLC25A38 was situated upstream of metastasis-related pathways, especially angiogenesis. Low expression of SLC25A38 promotes angiogenesis and metastasis, and identifies increased metastatic risk and worse survival in UM patients. This finding may further improve the accuracy of prognostic prediction for UM.

LSD1-Demethylated LINC01134 Confers Oxaliplatin Resistance Through SP1-Induced p62 Transcription in HCC.

BACKGROUND AND AIMS: Oxaliplatin (OXA) is one of the most common chemotherapeutics in advanced hepatocellular carcinoma (HCC), the resistance of which poses a big challenge. Long noncoding RNAs (lncRNAs) play vital roles in chemoresistance. Therefore, elucidating the underlying mechanisms and identifying predictive lncRNAs for OXA resistance is needed urgently. METHODS: RNA sequencing (RNA-seq) and fluorescence in situ hybridization (FISH) were used to investigate the OXA-resistant (OXA-R) lncRNAs. Survival analysis was performed to determine the clinical significance of homo sapiens long intergenic non-protein-coding RNA 1134 (LINC01134) and p62 expression. Luciferase, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and chromatin isolation by RNA purification (ChIRP) assays were used to explore the mechanisms by which LINC01134 regulates p62 expression. The effects of LINC01134/SP1/p62 axis on OXA resistance were evaluated using cell viability, apoptosis, and mitochondrial function and morphology analysis. Xenografts were used to estimate the in vivo regulation of OXA resistance by LINC01134/SP1/p62 axis. ChIP, cell viability, and xenograft assays were used to identify the demethylase for LINC01134 up-regulation in OXA resistance. RESULTS: LINC01134 was identified as one of the most up-regulated lncRNAs in OXA-R cells. Higher LINC01134 expression predicted poorer OXA therapeutic efficacy. LINC01134 activates anti-oxidative pathway through p62 by recruiting transcription factor SP1 to the p62 promoter. The LINC01134/SP1/p62 axis regulates OXA resistance by altering cell viability, apoptosis, and mitochondrial homeostasis both in vitro and in vivo. Furthermore, the demethylase, lysine specific demethylase 1 (LSD1) was responsible for LINC01134 up-regulation in OXA-R cells. In patients with HCC, LINC01134 expression was positively correlated with p62 and LSD1 expressions, whereas SP1 expression positively correlated with p62 expression. CONCLUSIONS: LSD1/LINC01134/SP1/p62 axis is critical for OXA resistance in HCC. Evaluating LINC01134 expression in HCC will be effective in predicting OXA efficacy. In treatment-naive patients, targeting the LINC01134/SP1/p62 axis may be a promising strategy to overcome OXA chemoresistance.

PTK2 promotes cancer stem cell traits in hepatocellular carcinoma by activating Wnt/ß-catenin signaling.

Emerging evidence indicates that cancer stem cells (CSCs) are involved in tumorigenesis, tumor recurrence, and therapeutic resistance in hepatocellular carcinoma (HCC). However, the mechanisms underlying HCC CSC regulation remain largely unknown. Here we report our analysis of 97 paraffin-embedded HCC tumor specimens. We found that protein tyrosine kinase 2 (PTK2) expression correlated with liver CSC marker expression, overall survival, and recurrence-free survival in HCC patients. Our results further showed that PTK2 activated Wnt/ß-catenin signaling by promoting nuclear accumulation of ß-catenin in HCC cells. In this manner, PTK2 activates CSC traits and drives tumorigenicity in HCC cells, leading to HCC recurrence and sorafenib resistance. Moreover, PTK2 expression was negatively correlated with its level of promoter methylation. PTK2 apparently acts as an oncogene by increasing CSC traits and tumorigenicity in HCC. The present data suggest that PTK2 may be a novel prognostic biomarker for HCC recurrence, and a therapeutic target for HCC treatment.

The risk variant rs884225 within EGFR impairs miR-103a-3p's anti-tumourigenic function in non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) status is the major determinant of non-small cell lung cancer (NSCLC) therapy selection. Studies have hinted that EGFR antibodies or tyrosine kinase inhibitors were beneficial in patients with EGFR mutation-negative but EGFR-overexpressing of NSCLC. However, the mechanisms underlying EGFR amplification and overexpression in NSCLC remain largely unknown. Here, we report that rs884225, a single nucleotide polymorphism in the EGFR 3'-terminal untranslated region, was significantly associated with EGFR expression level and contributed to NSCLC susceptibility. Mechanistically, the rs884225 C allele enhanced EGFR expression by altering the miR-103a-3p binding site, thus impairing miR-103a-3p's anti-tumourigenic function. As a tumour suppressor gene, miR-103a-3p expression correlated with overall and recurrence-free survival in NSCLC patients. Furthermore, miR-103a-3p inhibited growth and metastasis via effects on the KRAS pathway and epithelial-to-mesenchymal transition in EGFR wild-type NSCLC cell lines, respectively, which substantially reduced EGFR expression and activity. Thus, rs884225 may be a biomarker for NSCLC susceptibility, and miR-103a-3p may be a potential therapeutic target in NSCLC.

Preference of cancer patients and family members regarding delivery of bad news and differences in clinical practice among medical staff.

OBJECTIVES: To study the preferences of cancer patients and their families in way of being informed of their condition and, by comparing their preferences with the medical staff's clinical practices, explore the factors underlying the latter's preferences. METHODS: A survey was conducted with 216 cancer patients, 242 families, and 176 clinical staff members with the Medical Status Communication questionnaire (Simplified Chinese edition). RESULTS: The clinical staff scored lower than the cancer patients and their families in terms of the total score, way of communication, emotional support, and additional information (F = 16.134, p < .001; F = 28.604, p < .001; F = 13.839, p < .001; F = 16.745, p < .001). Factors underlying the medical staff's clinical practices included, as revealed by the multiple linear regression analysis, gender (p = .03), and willingness to improve the way of communication about cancer (p = .006). CONCLUSIONS: A gap existed between the medical staff's clinical practice and the preferences of the cancer patients and their families. The medical staff should receive adequate training in cancer communication skills and techniques for improvement in this respect. When designing training for skills in delivering bad news to cancer patients, the well-being of cancer patients and their families must be thoroughly considered, and patient demands for information should be satisfied in the context of the information explosion of the current age.

A nomogram-based immunoprofile predicts overall survival for previously untreated patients with esophageal squamous cell carcinoma after esophagectomy.

BACKGROUND: Immunoscore, as a prognostic tool defined to quantify in situ immune cell infiltrates, appears to be superior to the TNM staging system. In esophageal squamous cell carcinoma (ESCC), no immunoscore has been established; however, in situ tumor immunology is recognized as highly important. Our study aimed to construct a comprehensive immunoprofile for ESCC. METHODS: The infiltration of four immune cell types (CD8+/CD4+/Foxp3+/CD33+ cells), the expression of both inhibitory (PD-1/PD-L1/Tim-3/LAG-3) and stimulatory checkpoints (OX-40/ICOS), and IDO1 were evaluated by IHC staining and multi-color immunofluorescence in two independent cohorts (95 patients in the primary cohort and 55 patients in the validation cohort). The association with patients' overall survival was analyzed by the Kaplan-Meier method and the Cox model. Nomogram-based immunoprofile was established using the independent prognostic variables. To determine its predictive accuracy and discriminatory capacity, the C-index and calibration curve were calculated. RESULTS: Significant correlation of PD-L1 expression in tumor cells with PD-1+ T cell infiltration was found (P = 0.035), indicating the activation of the inhibitory PD-1/PD-L1 pathway in ESCC cases. More PD-L1+ ICs, Tim-3+ ICs and LAG-3+ ICs were found in the CD8-rich tumor microenvironment, which is in accordance with the feedback nature of immune system. After adjustment by TNM stage, four immune variables including the infiltration of CD8+/Foxp3+/CD33+ cells and the PD-L1 expression by tumor cells were selected to construct a prognostic nomogram. The calibration curves showed good accuracy of the nomogram for survival prediction. To overcome the complexity of applying a nomogram in a clinical setting, a simple immunoprofile was then established according to the points of each factor from the nomogram. Our immunoprofile model could separate same-stage patients into different risk subgroups, and showed superior accuracy for survival prediction than the TNM staging system based on the C-index calculation and ROC analysis. CONCLUSIONS: Our nomogram-based immunoprofile can provide more accurate prognosis prediction and is an important complement to the TNM staging system for operable ESCC patients.

miR-216a inhibits osteosarcoma cell proliferation, invasion and metastasis by targeting CDK14.

Osteosarcoma (OS) has emerged as the most common primary musculoskeletal malignant tumour affecting children and young adults. Cyclin-dependent kinases (CDKs) are closely associated with gene regulation in tumour biology. Accumulating evidence indicates that the aberrant function of CDK14 is involved in a broad spectrum of diseases and is associated with clinical outcomes. MicroRNAs (miRNAs) are crucial epigenetic regulators in the development of OS. However, the essential role of CDK14 and the molecular mechanisms by which miRNAs regulate CDK14 in the oncogenesis and progression of OS have not been fully elucidated. Here we found that CDK14 expression was closely associated with poor prognosis and overall survival of OS patients. Using dual-luciferase reporter assays, we also found that miR-216a inhibits CDK14 expression by binding to the 3'-untranslated region of CDK14. Overexpression of miR-216a significantly suppressed cell proliferation, migration and invasion in vivo and in vitro by inhibiting CDK14 production. Overexpression of CDK14 in the miR-216a-transfected OS cells effectively rescued the suppression of cell proliferation, migration and invasion caused by miR-216a. In addition, Kaplan-Meier analysis indicated that miR-216a expression predicted favourable clinical outcomes for OS patients. Moreover, miR-216a expression was downregulated in OS patients and was negatively associated with CDK14 expression. Overall, these data highlight the role of the miR-216a/CDK14 axis as a novel pleiotropic modulator and demonstrate the associated molecular mechanisms, thus suggesting the intriguing possibility that miR-216a activation and CDK14 inhibition may be novel and attractive therapeutic strategies for treating OS patients.

miR-30a-5p enhances paclitaxel sensitivity in non-small cell lung cancer through targeting BCL-2 expression.

Lung cancer remains the leading cause of cancer-related death worldwide. Paclitaxel, either as monotherapy or combined with other agents, is the standard treatment for advanced non-small cell lung cancer (NSCLC), the most common type of lung cancer. However, both de novo and acquired resistance against paclitaxel frequently occurs and represents a huge clinical problem. The underlying mechanisms remain poorly characterized. Here, by comparing microRNA (miRNA) expression levels using miRNA arrays, we observed differential expression of miR-30a-5p in two independent lung cancer cell pairs (paclitaxel-resistant vs paclitaxel-sensitive A549 cell lines). Overexpression of miR-30a-5p sensitizes NSCLC cells to paclitaxel both in vitro and in vivo. In addition, miR-30a-5p increases paclitaxel sensitivity by promoting chemotherapy-induced apoptosis via downregulating BCL-2, a key apoptosis regulator. High miR-30a-5p expression is positively correlated with enhanced responsiveness to paclitaxel and predicts a more favorable clinical outcome in NSCLC patients. Moreover, miR-30a-5p expression is negatively correlated with BCL-2 expression in NSCLC tissues. These data indicate that miR-30a-5p may be useful to treat paclitaxel-resistant lung cancer and may also provide a biomarker to predict paclitaxel responsiveness in lung cancer. KEY MESSAGES: BCL-2 is a novel direct target of miR-30a-5p. miR-30a-5p enhances NSCLC paclitaxel sensitivity in vitro and in vivo. miR-30a-5p sensitizes NSCLC cells to paclitaxel by inducing apoptosis through BCL-2 inhibition. miR-30a-5p negatively correlates with BCL-2 and predicts a favorable clinical outcome in NSCLC patients.

A signature motif in LIM proteins mediates binding to checkpoint proteins and increases tumour radiosensitivity.

Tumour radiotherapy resistance involves the cell cycle pathway. CDC25 phosphatases are key cell cycle regulators. However, how CDC25 activity is precisely controlled remains largely unknown. Here, we show that LIM domain-containing proteins, such as FHL1, increase inhibitory CDC25 phosphorylation by forming a complex with CHK2 and CDC25, and sequester CDC25 in the cytoplasm by forming another complex with 14-3-3 and CDC25, resulting in increased radioresistance in cancer cells. FHL1 expression, induced by ionizing irradiation in a SP1- and MLL1-dependent manner, positively correlates with radioresistance in cancer patients. We identify a cell-penetrating 11 amino-acid motif within LIM domains (eLIM) that is sufficient for binding CHK2 and CDC25, reducing the CHK2-CDC25 and CDC25-14-3-3 interaction and enhancing CDC25 activity and cancer radiosensitivity accompanied by mitotic catastrophe and apoptosis. Our results provide novel insight into molecular mechanisms underlying CDC25 activity regulation. LIM protein inhibition or use of eLIM may be new strategies for improving tumour radiosensitivity.

miR-105/Runx2 axis mediates FGF2-induced ADAMTS expression in osteoarthritis cartilage.

UNLABELLED: Fibroblast growth factor 2 (FGF2) plays an important role in the development of osteoarthritis (OA) through the regulation of cartilage degradation. However, the molecular mechanism underlying FGF2-induced OA is poorly characterized. MicroRNAs (miRNAs) maintain cartilage homeostasis. To examine whether FGF2 regulates OA through the modulation of miRNA, we screened potential miRNA molecules that could be regulated through FGF2 using microarray analysis. The results showed that microRNA-105 (miR-105) was significantly downregulated in chondrocytes stimulated with FGF2. Runt-related transcription factor 2 (Runx2), a key transcription factor involved in OA, has been identified as a novel potential target of miR-105. FGF2 suppressed miR-105 expression through the recruitment of the subunit of the nuclear factor kappa B transcription complex p65 to the miR-105 promoter. The knockdown of Runx2 mimicked the effect of miR-105 and abolished the ability of miR-105 to regulate the expression of a disintegrin-like and metalloproteinase with thrombospondin 4 (ADAMTS4), ADAMTS5, ADAMTS7 and ADAMTS12, both of which are responsible for the degradation of collagen 2A1 (COL2A1) and aggrecan (ACAN). miR-105 is also required for FGF2/p65-induced Runx2 activation and ADAMTS expression. Moreover, miR-105 expression was downregulated in OA patients and inversely correlated with the expression of Runx2, ADAMTS7 and ADAMTS12, which were upregulated in OA patients. These data highlight that the FGF2/p65/miR-105/Runx2/ADAMTS axis might play an important role in OA pathogenesis and that miR-105 might be a potential diagnostic target and useful strategy for OA treatment. KEY MESSAGE: Runx2 was identified as a novel direct target of miR-105. FGF2 inhibits miR-105 transcription through recruitment of p65 to miR-105 promoter. p65/miR-105 is essential for FGF2-mediated Runx2 and ADAMTS upregulation. miR-105 is downregulated in OA and inversely correlated with Runx2 expression.

[Construction of human Bcl-6 3'UTR reporter vector and expression vector and their functional assessment].

OBJECTIVE: To observe the direct regulation of miR-127 on Bcl-6 and the effect of Bcl-6 in rescuing miR-127-induced cell cycle and cell growth inhibition. METHODS: The 3'UTR and coding region of human bcl-6 gene were amplified by PCR and cloned into pcDNA3.0-Luc and pcDNA3.0-Flag vectors, respectively. Mutations were introduced into the seed sequences of the predicted miR-127 target sites within the Bcl-6 3'UTR using recombinant PCR. Luciferase assay was used to verify the direct targeted regulation of miR-127 on Bcl-6. In HepG2 cell models with overexpression or knockdown of miR-12, the changes of cell cycle and cell growth were investigated after transfection with the constructed vectors. RESULTS: The recombinant plasmids were successfully obtained as confirmed by double digestion and sequence identification. Luciferase assay showed that in 293T and HepG2 cells, miR-127 inhibited the activation of wild-type Bcl-6 3'UTR reporter vector but not mutated Bcl-6 3'UTR vector. Overexpression of miR-127 induced cell cycle arrest at G(2)/M phase and suppressed the growth of HepG2 cells, and these effects were reversed by Bcl-6 overexpression. CONCLUSION: We successfully cloned wild-type and mutated 3'UTR reporter vectors and expression vector of bcl-6 gene and confirmed their biological functions.

Improved synthesis and biological evaluation of Tc-99m radiolabeled AMO for miRNA imaging in tumor xenografts.

MicroRNAs (miRNAs) have been considered as important biomarkers for malignant tumors. In this study, we introduced an improved (99m)Tc labeling method for noninvasive visualization of overexpressed miRNAs in tumor-bearing mice. Anti-miRNA-21 oligonucleotide (AMO) with partial 2'-O-methyl and phosphorothioate modification was designed and chemically synthesized. After conjugated with NHS-MAG3, AMO was labeled with (99m)Tc. Optimization was made to shorten reaction time and to improve labeling efficiency. Labeling efficiency was 97%, and specific activity was 2.78 MBq/ng. During 12 h, (99m)Tc-AMO showed no significant degradation by gel electrophoresis. Its radiochemical purity was stable, between 95.8% and 99.1%. Further, (99m)Tc-AMO decreased the level of miR-21 and increased the expression of PTEN protein at cellular level, shown by qRT-PCR and Western blot. Fluorescent protein labeled AMO displayed specific distribution and good stability in tumor cells. After the administration in tumor-bearing mice, (99m)Tc-AMO showed more radioactive uptake in the miR-21 over-expressed tumors than scramble control. Biodistribution results further proved the significant difference of tumor uptake between (99m)Tc-AMO and (99m)Tc-control. Therefore, this study presents an improved method with shorten time to prepare a (99m)Tc radiolabeled AMO. In addition, it supports the role of (99m)Tc-AMO for noninvasive visualization of miR-21 in malignant tumors.

MiR-125a suppresses tumor growth, invasion and metastasis in cervical cancer by targeting STAT3.

MiR-125a has been characterized as a tumor suppressor in several cancers. However, the role of miR-125a in cervical cancer is unknown. In this study, we found the expression of miR-125a was downregulated in cervical cancer patients, and negatively correlated with the tumor size, FIGO stage, and preoperative metastasis. Kaplan-Meier analysis showed that miR-125a expression predicted favorable outcome for cervical cancer patients. Dual luciferase assays identified the STAT3 gene as a novel direct target of miR-125a. Functional studies showed that miR-125a overexpression significantly suppressed the growth, invasion and epithelial-mesenchymal transition (EMT) of cervical cancer cells both in vitro and in vivo via decreasing STAT3 expression. Moreover, miR-125a conferred to G2/M cell cycle arrest, accompanied by inhibition of several G2/M checkpoint proteins. Mechanistically, inactivation of miR-125a during cervical carcinogenesis was caused by HPV suppression of p53 expression. Clinically, STAT3, the expression of which, predicted poorer outcome, was inversely correlated with miR-125a in cervical cancer. These data highlight the importance of miR-125a in the cell proliferation and progression of cervical cancer, and indicate that miR-125a may be a useful therapeutic target for cervical cancer.

[FHL1 knockdown mediated by lentiviral shRNA promotes the growth of HeLa and HepG2 cells].

OBJECTIVE: To investigate the effect of four-and-a-half LIM domain 1 (FHL1) knockdown by lentiviral-mediated shRNA on the growth of HeLa and HepG2 cells. METHODS: pLenti-H1 FHL1 shRNA was cloned, and then transfered into HEK293T cells. The inhibitory effect of pLenti-H1 FHL1 shRNA on FHL1 gene was detected by Western blotting and real-time quantitative PCR (qRT-PCR). Lentivirus particles were packaged, added to HeLa and HepG2 cells, followed by puromycin treatment for 2-3 weeks to screen stable clones. The knockdown effect on FHL1 expression in these cells was checked by Western blotting and qRT-PCR. Cell growth and colony formation analysis were performed to investigate the effect of FHL1 down-regulation on tumor cell growth. Soft agar analysis was used to analyze its effect on anchorage-independent tumor cell growth. RESULTS: Western blotting and qRT-PCR revealed that the pLenti-H1 FHL1 shRNA apparently inhibited the expression of FHL1 gene. Cell growth and colony formation assay showed that the lentiviral-mediated shRNA for FHL1 gene significantly accelerated the tumor cell growth in HepG2 and HeLa cells. Soft agar analysis demonstrated that FHL1 shRNA increased the anchorage-independent growth of tumor cells. CONCLUSION: pLenti-H1 FHL1 shRNA could significantly accelerate tumor cell growth via inhibiting the expression of FHL1.

Hematopoietic pre-B cell leukemia transcription factor interacting protein is overexpressed in gastric cancer and promotes gastric cancer cell proliferation, migration, and invasion.

Hematopoietic pre-B cell leukemia transcription factor interacting protein (HPIP) has been shown to play an important role in the development and progression of some cancers. However, the role of HPIP in gastric cancer (GC) is unclear. Here, we show that HPIP is upregulated in most GC patients and promotes GC cell proliferation, migration, and invasion. In GC patients, HPIP positively associates with tumor size and nodal metastasis, and negatively associates with tumor differentiation. Hematopoietic pre-B cell leukemia transcription factor interacting protein increases GC cell proliferation through activation of G1 /S and G2 /M cell cycle transitions, accompanied by a marked increase of the positive cell cycle regulators, including cyclin D1, cyclin A, and cyclin B1. Hematopoietic pre-B cell leukemia transcription factor interacting protein enhances GC cell migration and invasion, and modulates epithelial-mesenchymal transition, which plays a key role in cancer cell migration and invasion. These data underscore the critical role of HPIP in GC cell proliferation and progression and suggest that HPIP inhibition may be a useful therapeutic strategy for GC treatment.

[myc-p62 fusion protein suppresses the phosphorylation of extracellular signal-regulated kinase].

OBJECTIVE: To construct a eukaryotic expression vector of human autophagy maker protein P62 labeled with myc tag, and detect its biological function. METHODS: Human P62 gene was amplified from human breast DNA library by PCR and cloned into pXJ-40-myc vector. HEK293T cells were transfected with the recombinant plasmid myc-p62. Western blotting was conducted to detect the fusion protein expression and the effect on the phosphorylation of extracellular signal-regulated kinase (ERK). RESULTS: Double enzyme identification and sequencing result showed that P62 eukaryotic expression vector labeled with myc tag was successfully constructed and the inserted fragment was correct. Western blotting indicated that the fusion protein was successfully expressed and was able to inhibit ERK phosphorylation. CONCLUSION: The eukaryotic expression vector of myc-p62 was successfully constructed and proved to have an inhibitory effect on ERK phosphorylation.

Metformin inhibits thyroid cancer cell growth, migration, and EMT through the mTOR pathway.

Mammalian target of rapamycin (mTOR) signaling pathways have been shown to be activated in thyroid cancer. Recent evidences have demonstrated that the antidiabetic agent metformin, an activator of 5'-AMP-activated protein kinase, can impair the proliferation and migration of cancer cells via inhibition of mTOR. However, the underlying mechanisms remain unclear. In this study, we show that metformin can inhibit mTOR pathway to impair growth and migration of the thyroid cancer cell lines. Cyclin D1 and c-Myc are important regulators of cancer cell growth, and we observed that treatment of thyroid cancer cells with metformin reduced c-Myc and cyclin D1 expression through suppression of mTOR and subsequent inhibition of P70S6K1 and 4E-BP1 phosphorylation. Metformin reduced epithelial to mesenchymal transition (EMT) in thyroid carcinoma cells. Moreover, metformin regulated expression of the EMT-related markers E-cadherin, N-cadherin, and Snail. Additionally, knockdown of TSC2, the upstream regulatory molecule of mTOR pathway, or treatment of rapamycin, the mTOR inhibitor, could abolish the effects of metformin to regulate thyroid cancer cell proliferation, migration, EMT, and mTOR pathway molecules. These results indicate that metformin can suppress the proliferation, migration, and EMT of thyroid cancer cell lines by inhibiting mTOR signaling. These findings suggest that metformin and its molecular targets may be useful in thyroid carcinoma therapy.

MiR-410 is overexpressed in liver and colorectal tumors and enhances tumor cell growth by silencing FHL1 via a direct/indirect mechanism.

FHL1 is an important tumor-suppressor that is downregulated in multiple tumors by unknown mechanisms. We demonstrated that miR-410 specifically targets the 3'UTR of FHL1. Furthermore, using DNA bisulfite modification and sequencing experiments, we demonstrated that the FHL1 promoter is hypermethylated in cancer cells. FHL1 methylation is increased upon miR-410 expression, suggesting that the regulation of FHL1 by miR-410 occurs by a dual mechanism. Using chromatin immunoprecipitation assays, we observed that miR-410 overexpression results in the increased binding of DNMT3A at the FHL1 promoter, which could explain how miR-410 regulates FHL1 methylation. Importantly, in vitro and in vivo results suggest that miR-410 may have oncogenic properties. Furthermore, both miR-410 and DNMT3A are upregulated in clinical human liver and colorectal tumors cancers. Our results suggest that miR-410 may function as an oncomiR and are consistent with its key function in regulating FHL1 in certain digestive system cancers.

miR-30a suppresses breast cancer cell proliferation and migration by targeting Eya2.

Eye absent (Eya) proteins are involved in cell fate determination in a broad spectrum of cells and tissues. Aberrant expression of Eya2 has been documented in a variety of cancers and correlates with clinical outcome. However, whether microRNAs (miRNAs) can regulate Eya2 expression remains unknown. Here, we show that miR-30a represses Eya2 expression by binding to the 3'-untranslated region of Eya2. Overexpression of Eya2 in miR-30a-transfected breast cancer cells effectively rescued the inhibition of cell proliferation and migration caused by miR-30a. Knockdown of Eya2 by small-interfering RNA (siRNA) in breast cancer cells mimicked the effect induced by miR-30a and abolished the ability of miR-30a to regulate breast cancer cell proliferation and migration. The miR-30a/Eya2 axis could regulate G1/S cell cycle progression, accompanied by the modulation of expression of cell cycle-related proteins, including cyclin A, cyclin D1, cyclin E, and c-Myc. Moreover, miR-30a expression was downregulated in breast cancer patients, and negatively correlated with Eya2, which was upregulated in breast cancer patients. These data suggest that the miR-30a/Eya2 axis may play an important role in breast cancer development and progression and that miR-30a activation or Eya2 inhibition may be a useful strategy for cancer treatment.